Heparin Bioconjugate with a Thermoresponsive Cationic Branched

Jun 16, 2007 - Ryosuke Iwai , Shota Kusakabe , Yasushi Nemoto , and Yasuhide Nakayama .... Kingo Uchida , Taiji Watanabe , Keiichi Kanda , Hitoshi Yak...
0 downloads 0 Views 307KB Size
Download PDF
8206

Langmuir 2007, 23, 8206-8211

Heparin Bioconjugate with a Thermoresponsive Cationic Branched Polymer: A Novel Aqueous Antithrombogenic Coating Material Yasuhide Nakayama,*,†,‡ Ryohei Okahashi,†,§ Ryosuke Iwai,†,‡,§ and Kingo Uchida§ Department of Bioengineering, AdVanced Medical Engineering Center, National CardioVascular Center Research Institute, and DiVision of Biotechnology and Macromolecular Chemistry, Faculty of Engineering, Hokkaido UniVersity, and Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku UniVersity ReceiVed February 5, 2007. In Final Form: May 6, 2007 With a view to reducing the thrombogenic potential of artificial blood-contact devices and natural tissues, we developed a novel aqueous antithrombogenic coating material, comprising a heparin bioconjugate that incorporated a thermoresponsive cationic polymer as a surfactant. The polymer was prepared by the sequential steps of initiatortransfer agent-terminator (iniferter)-based living radical photopolymerization of N-[3-(dimethylamino)propyl]acrylamide, followed by the polymerization of N-isopropylacrylamide from tetra(N,N-diethyldithiocarbamylmethyl)benzene as a multifunctional iniferter. The polymer obtained possessed four branched chains, each consisting of a cationic PDMAPAAm block (Mn: ca. 3000 g‚mol-1) forming an inner domain for heparin binding and a thermoresponsive PNIPAM block (Mn: ca. 6000 g‚mol-1) forming an outer domain for surface fixation; bioconjugation of the polymer with heparin occurred immediately upon simple mixing in an aqueous medium. Because the lower critical solution temperature of the heparin bioconjugate was approximately 35 °C, it could be coated from an aqueous solution at room temperature. The excellent adsorptivity and high durability of the coating below 37 °C was demonstrated on several generally used polymers by wettability measurement and surface chemical compositional analysis, and on collagen sheets and rat skin tissue by heparin staining. Blood coagulation was significantly prevented on the heparin bioconjugate-coated surfaces. The thermoresponsive bioconjugate developed therefore appeared to satisfy the initial requirements for a biocompatible aqueous coating material.

Introduction The glycosaminoglycan heparin displays potent anticoagulant activity when complexed with antithrombin III (ATIII). Consequently, heparin has gained long-term and widespread clinical use as an anticoagulant during extracorporeal circulation. Furthermore, various heparinization techniques have been proposed and developed for conferring aniticoagulant properties to the blood-contacting surfaces of extracorporeal and implantable devices, including stents and catheters.1-3 These techniques include surface physical mixing,4,5 coating with a surfactantheparin complex,6 surface derivatization through chemical bonding with or without a spacer arm,7.8 and hydrogel immobilization.9 When heparin bioconjugated with a long alkylated * To whom correspondence should be addressed: Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 5658565, Japan, Telephone: (+81) 6-6833-5012 (ex. 2624), Fax: (+81) 6-68728090, E-mail: [email protected]. † National Cardiovascular Center Research Institute. ‡ Division of Biotechnology and Macromolecular Chemistry, Hokkaido University. § Ryukoku University. (1) Lee, Y. K.; Park, J. H.; Moon, H. T.; Lee, D. Y.; Yun, J. H.; Byun, Y. Biomaterials 2007, 28, 1523-1530. (2) Du, Y. J.; Brash, J. L.; McClung, G.; Berry, L. R.; Klement, P.; Chan, A. K. C. J. Biomed. Mater. Res. 2007, 80A, 216-225. (3) Parlinson, R. J.; Demers, C. P.; Adel, J. G.; Levy, E. I.; Sauvageau, E.; Hanel, R. A.; Shaibani, A.; Guterman, L. N.; Hopkins, L. N.; Batjer, H. H.; Bendok, B. R. Neurosurgery 2006, 59, 812-821. (4) Lin, J. Y.; Nojiri, C.; Okano, T.; Kim, S. W. ASAIO Trans. 1987, 33, 602-605. (5) Goosen, M. F.; Sefton, M. V. J. Biomed. Mater. Res. 1979, 13, 347-364. (6) Leninger, R. I.; Cooper, C. W.; Fab, R. D.; Grode, G. A. Science 1966, 152, 1625-1626. (7) Yuan, S.; Cai, W.; Szakalas-Grats, G.; Kottke-Marchant, K.; Tweden, K.; Marchant, R. E. J. Appl. Biomater. 1995, 6, 259-266. (8) Olsson, P.; Sanchez, J.; Mollnes, T. E.; Riesenfeld, J. J. Biomater. Sci. Polym. Ed. 2000, 11, 1261-1273.

amine via ionic bonding was coated onto an extracorporeal membrane oxygenation (ECMO) system, the acquisition of high antithrombogenicity and durability characteristics conductive to prolonged continuous cardiopulmonary support were reported.10 Using this system, the gas exchange function was maintained for several months under nonheparinized conditions. The coating of the heparin bioconjugate was performed using its alcohol solution; however, using an alcohol medium, it was difficult to use the coating material for the surface heparinization of natural tissues. Nevertheless, by reducing the terminal end of heparin, a terminally alkylated heparin coating material, which could coat from its aqueous solution, was prepared.11 It was concluded that the heparin surfactant might be utilized to confer reproducible, short-term systemic antithrombogenicity on assembled extracortoreal circulatory devices or circuits that lack long-term durability. Poly(N-isopropylacrylamide) (PNIPAM) is one of the thermoresponsive polymers that precipitates in water above 32 °C but is water-soluble at room temperature.12 Because of this unique feature, PNIPAM has been utilized for thermoresponsive tissue culture dishes,13,14 drug delivery vehicles,15,16 hemostasis,17 and (9) Goosen, M. F.; Sefton, M. V. J. Biomed. Mater. Res. 1983, 17, 359-373. (10) Tatsumi, E.; Eyu, K.; Taenaka, Y.; Nakatani, T.; Baba, Y.; Masuzawa, T.; Wakisaka, Y.; Toda, K.; Miyazaki, K.; Takano, H. ASAIO J. 1995, 41, M557M560. (11) Matsuda, T.; Magoshi, T. Biomacromolecules 2001, 2, 1169-1177. (12) Heskins, M.; Guillet, J. E. J. Macromol. Sci. Chem. 1968, A2, 14411455. (13) Okajima, S.; Sakai, Y.; Yamaguchi, T. Langmuir 2005, 21, 4043-4049. (14) Yamato, M.; Utsumi, M.; Kushida, A.; Konno, C.; Kikuchi, A.; Okano, T. Tissue Eng. 2001, 7, 473-480. (15) Nakayama, M.; Okano, T.; Miyazaki, T.; Kohori, F.; Sakai, K.; Yokoyama, M. J. Controlled Release 2006, 115, 46-56. (16) Zhang, J. X.; Qin, L. Y.; Jin, Y.; Zhu, K. J. J. Biomed. Mater. Res., Part A 2006, 76, 773-780. (17) Ohya, S.; Sonoda, H.; Nakayama, Y.; Matsuda, T. Biomaterials 2005, 26, 655-659.

10.1021/la700323m CCC: $37.00 © 2007 American Chemical Society Published on Web 06/16/2007

Heparin Bioconjugate for Antithrombogenic Coating

Langmuir, Vol. 23, No. 15, 2007 8207

Figure 1. Chemical structure of a cationic thermoresponsive copolymer (PDMAPAAm-PNIPAM 4-branched block copolymer) as a surfactant and its synthetic route.

3D extracellular matrix materials.18,19 Previously, heparin bioconjugated with PNIPAM via terminal graft copolymerization was developed as a biocompatible, thermoresponsive coating material. 20 The material had a low critical solution temperature (LCST), ranging from approximately 34 to 37 °C, that was dependent on the length of the alkyl chain. Although a high molecular weight PNIPAM chain (>5 × 105 g‚mol-1) is required for the stable adsorption of heparin, the unique thermoresponsive feature of PNIPAM may be useful as a means for adsorptiondriven surface modification by facilitating both simple coating using an aqueous solution of its bioconjugate at room temperature and concomitant physical stabilization on substrate surfaces, including natural tissues or organs, at physiological temperatures. In this study, a novel surfactant for an aqueous antithrombogenic coating material, combining PNIPAM as a surface adsorption domain with a cationic polymer as a heparin-binding domain, was molecularly designed. For the effective surface immobilization of heparin, a star-shaped branching configuration, which was expected to confer strong multipoint adsorption, was incorporated into the surfactant structure. The precise preparation of the branched cationic PNIPAM as a surfactant using initiatortransfer agent-terminator (iniferter)-based living radical photopolymerization 21,22 is described below, followed by a description of the surface functionality and a preliminary characterization of the biological activity of the heparin bioconjugate. Experimental Section Materials. 1,2,4,5-Tetrakis(bromomethyl)benzene was obtained from Sigma-Aldrich (Milwaukee, WI). Sodium N,N-diethyldithiocarbamate, N-[3-(dimethylamino)propyl]acrylamide (DMAPAAm), and N-isopropylacrylamide (NIPAM) were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). Other chemical reagents were commercially obtained from Wako Pure Chemical Ind., Ltd. The DMAAm was distilled under reduced pressure, and the NIPAM (18) Ohya, S.; Nakayama, Y.; Matusda, T. J. Artif. Organs 2004, 7, 181-186. (19) Naito, H.; Takewa, Y.; Mizuno, T.; Ohya, S.; Nakayama, Y.; Tatsumi, E.; Kitamura, S.; Takano, H.; Taniguchi, S.; Taenaka, Y. ASAIO J. 2004, 50, 344-348. (20) Magoshi, T.; Ziani-Cherif, H.; Ohya, S.; Nakayama, Y.; Matsuda, T. Langmuir 2002, 18, 4862-4872. (21) Otsu, T.; Matsumoto, A. AdV. Polym. Sci. 1998, 136, 75-137. (22) Otsu, T.; Yoshida, M. Makromol. Chem. Rapid Commun. 1982, 3, 127132.

was recrystallized with methanol/hexane before use in order to remove the stabilizer. The other reagents were purified before use according to requirements. General Methods. 1H nuclear magnetic resonance (NMR) spectra were recorded in deuterium oxide (D2O) with a 300 MHz NMR spectrometer (Gemini-300; Varian, Palo Alto, CA) at room temperature. Gel permeation chromatography (GPC) analyses with N,Ndimethylformamide as a solvent were carried out with an HPLC8020 instrument (Tosoh, Tokyo, Japan) using Tosoh TSKgel R-3000 and R-5000 columns. The columns were calibrated with narrow weight distribution poly(ethylene glycol) standards (Tosoh). Synthesis of Thermoresponsive Cationic Polymer. The thermoresponsive, cationic polymer, 1,2,4,5-tetrakis(N,N-diethyldithiocarbamyl(poly(N-[3-(dimethylamino)propyl]acrylamide)-block-poly(N-isopropylacrylamide))benzene (PDMAPAAm-PNIPAM) 4branched block copolymer, was synthesized by iniferter-based living radical photopolymerization from 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl)benzene as a multifunctional iniferter with DMAPAAm and NIPAM as cationic and thermoresponsive monomers, respectively. The detailed synthetic procedure is described in the following sections (Figure 1). Synthesis of Multifunctional Iniferter. Sodium N,N-diethyldithiocarbamate (12 g, 53.3 mmol) was added to 100 mL of an ethanol solution of 1,2,4,5-tetrakis(bromomethyl)benzene (2 g, 4.45 mmol). After stirring the solution at room temperature for 4 d, a solid was obtained by filtration. This solid was dissolved in 40 mL chloroform and washed with deionized water (40 mL × 3). The organic layer was dried using MgSO4 and condensed to give 2.79 g (88% yield) of the 4-functional iniferter, 1,2,4,5-tetrakis(N,Ndiethyldithiocarbamylmethyl)benzene. The observed 1H NMR chemical shifts (ppm) were as follows: δ 1.30-1.26 (t, 24H, -CH2CH3), 3.77-3.69 (q, 8H, -N-CH2-), 4.07-3.99 (q, 8H, -N-CH2-), 4.57 (s, 8H, Ar-CH2S), 7.49 (s, 2H, Ar-H). Synthesis of PDMAPAAm 4-Branched Polymer. DMAPAAm (0.86 g, 5.5 mmol) was added to 6 mL of a benzene solution of the multifunctional iniferter (72 mg, 0.1 mmol); this was diluted to a final volume of 20 mL with methanol and placed into a 50 mL quartz crystal tube. After bubbling with dry N2 gas for 5 min to exclude air, the solution was irradiated for 90 min with a 200 W highpressure mercury lamp (SPOT CURE; USHIO, Tokyo, Japan) under an N2 atmosphere at room temperature. The light intensity was set to 1 mW/cm2 at a wavelength of 250 nm (UVR-1; TOPCON, Tokyo, Japan). The reaction mixture was concentrated and adjusted to an appropriate concentration in order to facilitate purification by precipitation in a large volume of ether. Reprecipitation was carried

8208 Langmuir, Vol. 23, No. 15, 2007 out in a methanol-ether system. The final precipitate was dried under vacuum to yield a PDMAPAAm 4-branched polymer (conversion, 13%). The molecular weight (Mn) as determined by GPC analysis was 3700 g‚mol-1. 1H NMR: δ 1.7-1.5 (br, 3H, -CH2-CH- and -CH2-CH2-CH2-), 2.0-1.8 (br, 1H, -CH-CO), 2.22.1 (br, 6H, -N-CH3), 2.4-2.2 (br, 2H, -CH2-N(CH3)2), 3.2-3.0 (br, 2H, -NH-CH2-), 7.8-7.4 (br, 1H, -NH). Synthesis of PDMAPAAm-PNIPAM 4-Branched Block Copolymer. A methanol solution (20 mL) of the obtained PDMAPAAm 4-branched polymer (650 mg, 0.18 mmol) and NIPAM (1.24 g, 11 mmol) was placed into a 50 mL quartz crystal tube and deoxygenated by bubbling with dry N2 gas for 5 min. The solution was then irradiated for 90 min under the above-mentioned conditions. The reaction mixture was concentrated and precipitated in 400 mL of ether. Reprecipitation was carried out in a methanol-ether system. The final precipitate was dried under vacuum to yield the PDMAPAAmPDMAAm 4-branched block copolymer (conversion, 19%). The Mn as determined by GPC analysis was 9300 g‚mol-1. 1H NMR: δ 1.1-1.2 (br, -CH(CH3)2 of NIPAM), 1.5-1.8 (br, -CH2CH- and -CH2CH2CH2- of PDMAPAAm), 2.2 (br, -N(CH3)2 of PDMAPAAm), 2.3-2.4 (br, -CH2N(CH3)2 of DMAPAAm), 3.1-3.4 (br, -NHCH2 of PDMAAm), 4.0 (br, -NHCH2(CH3)2 of NIPAM). Preparation of Heparin Bioconjugate with Thermoresponsive Cationic Polymer. The heparin bioconjugate was prepared by the addition of heparin (10-20 mg) to 2 mL of an aqueous solution of the PDMAPAAm-PNIPAM 4-branched copolymer (20 mg) at room temperature. The size of the heparin bioconjugate was measured with dynamic light scattering (DLS) using an ELS-8000 system (Otuska Co., Osaka, Japan) equipped with a 10 mW He-Ne laser. Surface Characterization. The chemical composition of the outermost surface layer was determined by X-ray photoelectron spectroscopy (XPS 3400; Shimadzu Co., Kyoto, Japan) using a magnesium anode (Mg KR radiation) at room temperature under 5 × 10-6 Torr (10 kV, 20 mA) at a takeoff angle of 90°, where the takeoff angle is defined as the angle between the sample surface and the electron optics of the energy analyzer. The surface wettability was evaluated by measuring the static contact angles (advancing and receding) toward pure water using the sessile drop method with a contact angle meter (CA-V 200; Kyowa Kaimenkagaku Co., Ltd., Tokyo, Japan).

Results Synthesis of Block Copolymers. The PDMAPAAm-PNIPAM 4-branched block copolymer, which was designed as an adsorbent for the surface immobilization of heparin, was synthesized as depicted in the reaction scheme shown in Figure 1. In this reaction, iniferter-based living radical photopolymerization, in which the chain length can be easily controlled by changing the irradiation (time or light intensity) conditions and solution (iniferter or monomer concentration) conditions, was repeated for two different monomers of DMAPAAm and NIPAM. Initially, the polymerization of a cationic monomer (DMAPAAm) from a multifunctional iniferterssynthesized by the dithiocarbamylation of 1,2,4,5-tetrakis(bromomethyl)benzenes was performed using a different monomer concentration in order to obtain PDMAPAAm 4-branched polymers. Upon 90 min of UV light irradiation of DMAPAAm, a GPC trace showing an Mn of approximately 3700 g‚mol-1 was newly observed coincidentally with the disappearance of the initial GPC trace of the iniferter showing an Mn of 720 g‚mol-1 (Figure 2); this indicated the production of a PDMAPAAm cationic polymer with four branches. Second, the photopolymerization of a thermoresponsive monomer, NIPAM, from the obtained PDMAPAAm 4-branched polymers was conducted under similar polymerization conditions. Upon UV irradiation in the presence of NIPAM, the GPC curve was completely shifted toward a high molecular weight region (Figure 2). The polydispersity, however, was only slightly altered,

Nakayama et al.

Figure 2. Gel permeation chromatography elution curves, number average molecular weighs (Mn), and polydispersities (Mw/Mn) before (PDMAPAAm 4-branched polymer) and after block copolymerization (PDMAPAAm-PNIPAM 4-branched block copolymer).

Figure 3. Thermoresponsive change in transmittance of the surfactant (PDMAPAAm-PNIPAM 4-branched block copolymer) or heparin bioconjugate with the surfactant. Concentration of surfactant: 10 mg‚mL-1. Concentration of heparin: 5 mg‚mL-1. Heating rate: 0.5 °C‚min-1.

with the value remaining below 2. The molecular weight, which was determined by GPC, increased to approximately 9300 g‚mol-1. In addition, the 1H NMR spectra revealed that the peaks of the methyl protons at 1.1-1.2 ppm and N-methylene protons at approximately 4.0 ppm, all of which originated from the NIPAM unit, were newly detected in the obtained polymers. Therefore, it can be inferred that graft copolymerization occurred at the terminals of each chain of the PDMAPAAm to produce the block copolymer with four PDMAPAAm-PNIPAM block chains, comprising a PDMAPAAm and a PNIPAM block of approximately 750 and 1500 g‚mol-1, respectively. Preparation and Characterization of Heparin Bioconjugate. The resultant PDMAPAAm-PNIPAM 4-branched block copolymer was a white solid that dissolved readily in water at room temperature. When an aqueous solution of the block copolymer was mixed with an aqueous solution of heparin, an increase in the DLS intensity was immediately observed, indicating the formation of polymer/heparin complexessthe heparin bioconjugate. The size of the heparin bioconjugates obtained from 1% of the block copolymer and 0.5% of the heparin was approximately 15 nm in mean cumulant diameter. The heparin bioconjugate solution was precipitated in the physiological temperature range similar to the block copolymer solution (Figure 3). The equilibrium transmittance and LCST were approximately 50% and 36 °C, respectively, for the polymer solution, and approximately 55% and 35 °C for the heparin bioconjugate solution. The particle size of the heparin bioconjugate precipitated

Heparin Bioconjugate for Antithrombogenic Coating

Langmuir, Vol. 23, No. 15, 2007 8209

Table 1. Surface Chemical Composition Change of the PET Film Before and After Coating of the Heparin Bioconjugate elemental ratioc surface

O/C

N/C

S/C

PET + polymer PET + (polymer + heparin)b heparin

0.33 (0.4) 0.12 (0.14) 0.28 0.86 (0.89)

0 (0) 0.14 (0.20) 0.04 0.04 (0.08)

0 (0) 0 (0) 0.01 0.08 (0.07)

PETa

a PET: poly(ethylene terephthalate). b Weight ratio of polymer/heparin ) 2. c Determined by electron spectroscopy for chemical analysis.

Upon staining the coating surfaces with a dilute aqueous solution of toluidine blue after washing with water at 37 °C, only the coated regions on both materials were stained blue. This indicated that heparin, which can combine ionically with the dye, was fixed on the natural polymer or natural tissue surfaces in a manner similar to the synthetic polymer surfaces. Antithrombic Activity. It was of interest to observe how the heparin bioconjugate-adhered surface improved the blood compatibility and guaranteed the antithrombogenic potential. The degree of coagulation of whole rat blood was preliminary evaluated on a PET film surface with or without a coating of the heparin bioconjugate. Figure 5C,D shows photographs of the time-dependent changes in blood coagulation. As expected, there was no blood coagulation on the coated surface for up to 20 min, whereas blood gradually coagulated on the untreated PET surface. A potent anticoagulant activity was therefore obtained by heparin surface fixation.

Discussion

Figure 4. Receding water contact angles of heparin bioconjugatecoated polymeric surfaces after washing with water at 50 °C and then 10 °C.

at 37 °C was approximately 550 nm in cumulant diameter, which was notably different from the value (approximately 350 nm) obtained for an aqueous solution containing only the block copolymer. Therefore, upon heating to 37 °C, the block copolymer could be precipitated with the immobilization of heparin. Adhesivity of Heparin Bioconjugate. A poly(ethylene terephthalate) (PET) surface was coated with a mixed aqueous solution of the PDMAPAAm-PNIPAM 4-branched block copolymer (1%) and heparin (0.5%), subsequently air-dried, and then washed with water at either 10 or 50 °C. After washing at 50 °C, N1s and S2p signals in the XPS spectra were newly detected on the coating surface. The N/C and S/C elemental ratios, determined from the peak areas of the C1s, N1s, and S2p signals, were 0.04 and 0.01, respectively (Table 1). This indicates that the heparin bioconjugate was adhered onto the PET surface. The contact angle measurements indicated that, upon coating with the heparin bioconjugate, all the polymeric films examineds silicone (Si), polyethylene (PE), polystyrene (PST), and PETs became highly wettable (Figure 4). With the exception of the Si film, the receding water contact angle was less than 10°. For all the polymers, the high wettability remained almost unchanged after washing with water at 50 °C and also for PET and PST after washing at 10 °C. However, for PE and Si, partial delamination occurred after washing with water at 10 °C. On the basis of these observations, it was considered that the heparin bioconjugate displays strong affinity for the PST and PET surfaces. The low water contact angle on the PET surface coated with heparin bioconjugate remained after a 1 week soaking in water at 37 °C, indicating the high durability of the heparin bioconjugate. These wettability and XPS data led us to conclude that the heparin bioconjugate could be adhered onto polymeric surfaces with high reproducibility. Furthermore, the heparin bioconjugate was partly coated on collagen film (Figure 5A) and rat skin surfaces (Figure 5B).

In this study, we designed a unique surfactant material for the effective surface immobilization of heparin. As shown in Figure 1, the material comprised four AB-type blocked branches, each incorporating two different chemical entities: a cationic polymer block forming an inner domain and a thermoresponsive polymer block forming an outer domain. The control of the block chain lengths was realized by iniferter-based radical photopolymerization, which was originally developed by Otsu et al. in the early 1980s.21,22 The unique feature of this iniferter polymerization is that it proceeds in a quasi-living polymerization manner, in which “active” and “dormant” propagating chain ends are reversibly equilibrated under UV irradiation. Depending on the careful selection of appropriate reaction conditions, this enables either minimal transfer or termination reactions. In our previous studies, we developed iniferter-based, graft-polymerized surfaces23,24 and block copolymers as surface coatings,25,26 both of which were designed to realize biocompatible surfaces. Therefore, chain length and block ratio in the surfactant can be easily controlled by the iniferter polymerization. The chain lengths of the cationic and thermoresponsive blocks in the surfactant were set to approximately 3000 and 6000 g‚mol-1, respectively, as a prototype model compound (Figure 1). Upon mixing with heparin in an aqueous medium at room temperature, the surfactant immediately bioconjugated with heparin to produce nanoparticles of approximately 15 nm in diameter as illustrated in Figure 6. The surfactant possessed a cationic character due to the cationic nature of the poly(N,N-dimethylaminopropylacrylamide) polymer. In contrast, heparin has an anionic character by virtue of its sulfonic and carboxylic groups. Therefore, the resulting particles were polyion complexes formed by electrostatic interaction. Upon heating to 37 °C, the particles aggregated to form larger particles of approximately 550 nm in diameter, with this aggregation resulting from the conversion of the hydrophilic PNIPAM chains to hydrophobic chains at temperatures above the LCST. As illustrated in Figure 6, the resultant hydrophobic aggregates from the heparin bioconjugates could be stably adsorbed onto the surfaces of several hydrophobic polymers, including Si, PET, PST, and PE, all of which are routinely employed as materials (23) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 8622-8630. (24) Higashi, J.; Nakayama, Y.; Marchant, R. E.; Matsuda, T. Langmuir 1999, 15, 2080-2088. (25) Nakayama, Y.; Miyamura, M.; Hirano, Y.; Goto, K.; Matsuda, T. Biomaterials 1999, 20, 963-970. (26) Matsuda, T.; Nagase, J.; Ghoda, A.; Hirano, Y.; Kidoaki, S.; Nakayama, Y. Biomaterials 2003, 24, 4517-4527.

8210 Langmuir, Vol. 23, No. 15, 2007

Nakayama et al.

Figure 5. Photographs of collagen film (A) and rat skin (B) after partly coating with the heparin bioconjugate, washing with water at 37 °C, and then staining with an aqueous toluidine blue solution (0.1%). The coated areas as indicated by the arrows were strongly stained blue, indicating the heparin bioconjugate was adsorbed onto the naturally occurring polymer or tissue. Photographs of poly(ethylene terephthalate) (PET) films before (C) and after (D) heparin bioconjugate coating, on which whole blood was applied to the center of each surface, followed by washing with phosphate-buffered saline solution at 37 °C after a predetermined period of incubation at 37 °C.

Figure 6. Proposed structure of the heparin bioconjugate, which is a polyion complex of approximately 15 nm in diameter, and a representation of a heparin bioconjugate-coated surface.

for medical devices, which was clearly demonstrated by XPS measurement (Table 1). Therefore, the surface modification by a simple coating procedure with the heparin bioconjugate can be applied for almost all polymeric medical devices, for example, extracorporeal circulation, for the reduction of the systemic inflammatory response during cardiopulmonary bypass. It is considered that the coating, which can be applied in the form of an aqueous solution with no risk of damage to the device surface, will be particularly useful for drug-eluting stents (DESs), which have become the most effective devices of interventional treatment for coronary artery disease.27-30 However, safety concerns (27) Fattori, R.; Piva, T. Lancet 2003, 361, 247-249.

regarding the thrombogenicity of these materials remain to be addressed, since although the polymeric coating materials can control the release rates of incorporated drugs, their biocompatibilities have yet to be determined.31,32 Many approaches have (28) Lofina, E.; Haager, P. K.; Radke, P. W.; Langenberg, R.; Blindt, R.; Ortlepp, J.; Kuhl, H.; Hanrath, P.; Hoffmann, R. Catheter CardioVasc. InterV. 2005, 64, 28-34. (29) Nakayama, Y.; Ji-Youn, K.; Nishi, S.; Ueno, H.; Matsuda, T. J. Biomed. Mater. Res. 2001, 57, 559-566. (30) Nakayama, Y.; Nishi, S.; Ishibashi-Ueda, H. CardioVac. Radiat. Med. 2003, 4, 77-82. (31) Rogers, C. D. ReV. CardioVasc. Med. 2004, 5 (Suppl 2) S9-S15. (32) Ranade, S. V.; Miller, K. M.; Richard, R. E.; Chan, A. K.; Allen, M. J.; Helmus, M. N. J. Biomed. Mater. Res., Part A 2004, 71, 625-634.

Heparin Bioconjugate for Antithrombogenic Coating

Langmuir, Vol. 23, No. 15, 2007 8211

been used to generate a modified stent surface by chemically grafting or physically anchoring hydrophilic polymers such as heparin33-35 and phosphorylcholine-containing polymers.36 However, during the coating process, leakage of the drug impregnated in the polymeric matrix covering the stent strut may occur, since almost all drugs used are water-insoluble. Interestingly, since the heparin bioconjugate can be applied to biopolymers such as collagen film and natural tissues (represented in this study by rat skin, Figure 5), it will contribute to enhancing the reliability of implantations involving bloodcontacting tissues, for example, in bypass treatment during which injured coronary arteries are replaced with homografts. Therefore, our coating material will also undoubtedly be effective in implanting recently developed tissue-engineered tissues or organs in Vitro37,38 or in ViVo39-41 with the requisite antithrombogenic properties, which are expected to be the next generation of implantable materials, designed to overcome contemporary clinical difficulties.

In our previous study, we demonstrated that for adequate surface immobilization an extremely high molecular weigh PNIPAM chain of approximately >5 × 105 g‚mol-1 was required for heparin bioconjugates comprising a single PNIPAM chain.20 This amount was approximately 30 times greater than that of heparin. One-point bonding between heparin and PNIPAM was demonstrated to be an ineffective technique for surface immobilization. The novel surfactant material developed in the present study displayed the capacity of multipoint binding to heparin. In addition, the material had four-point adhesion sites to the substrate surface. As a consequence, a quantity of the polymeric material only twice that of heparin was effective for the surface immobilization of heparin. Moreover, as was demonstrated by the alkylated heparin, the multipoint binding of PNIPAM did not diminish the anticoagulant activity of heparin.10

(33) Mehran, R.; Aymong, E. D.; Ashby, D. T.; Fischell, T.; Whitworth, H Jr.; Siegel, R.; Thomas, W.; Wong, S. C.; Narasimaish, R.; Lansky, A. J.; Leon, M. B. Circulation 2003, 108, 1078-1083. (34) Serruys, P. W.; Emanuelsson, H.; van der Giessen, W.; Lunn, A. C.; Kiemeney, F.; Macaya, C.; Rutsch, W.; Heydrickx, G.; Suryapranta, H.; Legrand, V.; Goy, J. J.; Materne, P.; Bonnier, H.; Morice, M. C.; Fajadet, J.; Belardi, J.; Colombo, A.; Garcia, E.; Ruygrok, P.; de Jaegere, P.; Morel, M. A. Circulation 1996, 93, 412-422. (35) Faxon, D. P.; Spiro, T. E.; Minor, S.; Cote, G.; Douglas, J.; Gottlieb, R.; Califf, R.; Dorosti, K.; Topol, E.; Gordon, J. B. Circulation 1994, 90, 908-914. (36) Whelan, D. M.; van der Giessen, W. J.; Krabbendam, S. C.; van Vliet, E. A.; Verdouw, P. D.; Serruys, P. W.; van Beusekom, H. M. Heart 2000, 83, 338-345. (37) Poh, M.; Boyer, M.; Solan, A.; Dahl, S. L. M.; Pedrotty, D.; Banik, S. S. R.; McKee, J. A.; Klinger, R. Y.; Counter, C. M.; Niklason, L. E. Lancet 2005, 365, 2122-2124. (38) Niklason, L. E.; Gao, J.; Abbott, W. M.; Hirschi, K. K.; Houser, S.; Marini, R.; Langer, R. Science 1999, 284, 489-493. (39) Nakayama, Y.; Ishibashi-Ueda, H.; Takamizawa, K. Cell Transplant. 2004, 13, 439-449. (40) Sakai, O.; Kanda, K.; Takamizawa, K.; Ishibashi-Ueda, H.; Yaku, H.; Nakayama, Y. J. Biomed. Mater. Res., Part A, in press. (41) Hayashida, K.; Kanda, K.; Yaku, H.; Ando, J.; Nakayama, Y. J. Thorac. CardioVasc. Surg., in press.

The thermoresponsive bioconjugated material composed of a branched block polymer and heparin displayed a unique feature and appeared to meet the initial requirements for a biocompatible aqueous coating material, even though the developed material was primarily designed as a prototype model. Our understanding of the functionality of the bioconjugate will be advanced by the establishment of the molecular weight and by optimization of the surfactant polymer/heparin mixing ratio. In addition, the ideal branching number will be also determinedsa higher branching number will be more effective for surface binding. Our future research will initially be directed toward fine-tuning the composition of the surfactant material. We will then proceed to verify the long-term in ViVo biocompatible performance, including blood compatibility and toxicological safety, of the heparin bioconjugate.

Conclusion

LA700323M